Lane 1–4 reaction mixtures stopped after

0 s, 5, 15 and 30 min after addition of thrombin The electrophoretic patterns of fibrinogen under reducing conditions show the bands corresponding to Aα, Bβ and γ chains in the structure of this protein. Thrombin action on fibrinogen resulted in the disappearance of Aα and γ chains and appearance of additional bands corresponding to γ–γ chains, as well as high molecular weight α-polymers on the top of the gel (Fig. 2a). Preincubation of thrombin with cyanidin (2.5 μM) or quercetin (15 μM) significantly inhibited the formation of γ–γ chains and α-polymers, and inhibited the decay of bands corresponding selleck products to Aα and γ chains (Fig. 2b, c). The thrombin preincubation with cyanidin and with quercetin at IC50 concentration of amidolytic inhibition (0.25 μM for cyanidin and 1.5 μM for quercetin respectively) also inhibited the formation of γ–γ chains and α-polymers. However, after 15 min of the experiment, these bands corresponding to γ–γ chains and α-polymers appeared, while loss of bands corresponding to Aα and γ chains scarcely after 30 min was observed (Fig. 3b, c). SDS-PAGE

of fg treated with thrombin preincubated with silybin showed that this polyphenolic compound slightly reduced the formation of γ–γ chains and α-polymers at concentration of the compound of 250 μM. After 15 min, the electrophoretic pattern was similar to the control (Fig. 2d). In the Y-27632 ic50 electrophoresis of fg treated with thrombin preincubated with cyanin, (+)-catechin or (−)-epicatechin, no changes were observed (Fig. 2e–g). Fig. 3 The effect of polyphenolic compounds [cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin] on the thrombin-induced platelet aggregation. Thrombin was preincubated with polyphenols

at 37 °C for 10 min. Thrombin-catalyzed platelet aggregation was monitored for 10 min in the dual-channel Chrono-log aggregometer. The results are expressed as % of aggregation in comparison to the control samples (thrombin without tested compounds). Data represent mean ± SD of eight independent experiments done in duplicate The exposure of thrombin to cyanidin or quercetin resulted in dose-dependent decrease of the ability of thrombin to induce platelets aggregation. Cyanidin at a concentration of 5 μM reduced aggregation to 10 % of control, Montelukast Sodium while quercetin at a concentration of 50 μM reduced platelets aggregation to 4 % (Fig. 3a, b). Silybin effect on thrombin ability to induce platelet aggregation was also observed, but was much weaker when compared with cyanidin and quercetin, and at the concentration of 1,000 μM the aggregation reached 43 % of the control (Fig. 3c). Cyanin, (+)-catechin and (−)-epicatechin added to thrombin had no effect on thrombin ability to stimulate platelets aggregation (Fig. 3d–f). BIAcore analyses The sensorgrams obtained in BIAcore analyses (Fig.